Male FVB Mice Research Articles

Role of P-glycoprotein in Regulating the Efficacy, Toxicity and Pharmacokinetics of Yunaconitine.

Yunaconitine (YAC) is a hidden toxin that greatly threatens the life safety of patients who are prescribed herbal medicines containing Aconitum species; however, its underlying mechanism remains unclear. The objective of this study is to elucidate the functions of P-glycoprotein (P-gp) in regulating the efficacy, toxicity, and pharmacokinetics of YAC. The efflux function of P-gp on YAC was explored by using Caco-2 monolayers in combination with the P-gp inhibitor verapamil. The impact of P-gp on regulating the analgesic and anti-inflammatory effects, acute toxicity, tissue distribution, and pharmacokinetics of YAC was determined via male Mdr1a gene knocked-out mice and wild-type FVB mice. The presence of verapamil significantly decreased the efflux ratio of YAC from 20.41 to 1.07 in Caco- 2 monolayers (P < 0.05). Moreover, oral administration of 0.07 and 0.14 mg/kg YAC resulted in a notable decrease in writhing times in Mdr1a-/- mice by 23.53% and 49.27%, respectively, compared to wild-type FVB mice (P < 0.05). Additionally, the deficiency of P-gp remarkably decreased the half-lethal dose (LD50) of YAC from 2.13 to 0.24 mg/kg (P < 0.05). Moreover, the concentrations of YAC in the tissues of Mdr1a-/- mice were statistically higher than those in wild-type FVB mice (P < 0.05). Particularly, the brain accumulation of YAC in Mdr1a-/- mice significantly increased by 12- and 19-fold, respectively, after oral administration for 30 and 120 min, when compared to wild-type FVB mice (P < 0.05). There were no significant differences in the pharmacokinetic characteristics of YAC between Mdr1a-/- and wild-type FVB mice. YAC is a sensitive substrate of P-gp. The absence of P-gp enhances the analgesic effect and toxicity of YAC by upregulating its brain accumulation. Co-administration with a P-gp inhibitor may lead to severe YAC poisoning.

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.

Myeloid-derived suppressor cells attenuate the antitumor efficacy of radiopharmaceutical therapy using 90Y-NM600 in combination with androgen deprivation therapy in murine prostate tumors

RationaleAndrogen deprivation therapy (ADT) is pivotal in treating recurrent prostate cancer and is often combined with external beam radiation therapy (EBRT) for localized disease. However, for metastatic castration-resistant prostate cancer,...

Abstract 17462: Angiotensin II-Mediated Suppression of Skeletal Muscle Autophagy via mTOR and FoxO3a Pathways

Background: Chronic skeletal muscle (SK.M) atrophy, cachexia, occurs in diseases such as congestive heart failure (CHF), which is associated with high levels of angiotensin II (Ang-II). We have shown that Ang-II infusion reproduces major aspects of CHF-induced SK.M atrophy and causes mitochondrial (MT) damage and dysfunction in SK.M. Ang-II also inhibits autophagy/mitophagy, and a combination of these effects likely causes accumulation of damaged MT, leading to SK.M atrophy. Hypothesis: Ang-II infusion suppresses autophagy in SK.M via activation of mTOR and inhibition of FoxO3a. Aim: To understand the molecular mechanisms whereby Ang-II inhibits autophagy/mitophagy in SK.M. Methods: 12 weeks old male FVB mice were infused with Ang-II (1.0 μg·kg -1 ·min -1 ) or saline via osmotic minipumps (ALZET 1007D) for 7 days (N=6). Expression of autophagic markers and FoxO3a target genes were analyzed in tibialis anterior muscles by western blotting (at d1 &amp; d7) and qPCR (at d4), respectively. Colchicine treatment was included in the analyses to assess autophagic flux in vivo . For statistical analysis Two-way ANOVA and t-test were used. Results: In the presence of colchicine, LC3 lipidation was decreased in Ang-II infused mice, indicating that Ang-II inhibits autophagy at the autophagosome formation step. Furthermore, Ang-II increased p62 and PINK1 and decreased BNIP3, suggesting that Ang-II-mediated reduction of autophagy causes disruption of damaged MT clearance via mitophagy. Mechanistically, Ang-II likely inhibits autophagosome formation via two pathways, mTOR and FoxO3a: Ang-II increased phosphorylation of mTOR at Ser2448 (p&lt;0.005) and its downstream target 4E-BP1 at Thr37/46 (p&lt;0.0005). Lastly, Ang-II increased phosphorylation of FoxO3a at Thr32 (p&lt;0.0005), associated with reduced mRNA expression of FoxO3a target genes, such as LC3B and BNIP3 (24% and 46% reductions, respectively). Conclusion: Ang-II inhibits autophagosome formation in SK.M via mTOR activation and FoxO3a inhibition. Suppression of autophagy via these pathways likely disrupts clearance of damaged MT, leading to SK.M atrophy. These results provide insights for future development of therapies for CHF-induced cachexia by targeting mTOR and FoxO3a pathways to restore autophagy.

Mechanism of Social Stress-Related Erectile Dysfunction in Mice: Impaired Parasympathetic Neurotransmission and Ketamine.

This study aimed to investigate the mechanism underlying social stress (SS)-induced erectile dysfunction (ED) and evaluate the effects of a single subanesthetic dose of ketamine on SS-related ED. Male FVB mice were exposed to retired male C57BL/6 mice for 60 min daily over a 4-week period. In the third week, these FVB mice received intraperitoneal injections of either saline (SSS group) or ketamine (SSK group). Erectile function was assessed by measuring the intracavernosal pressure (ICP) during electrical stimulation of the major pelvic ganglia. Corpus cavernosum (CC) strips were utilized for wire myography to assess their reactivity. Both SSS and SSK mice exhibited significantly lower ICP in response to electrical stimulation than control mice. SS mice showed increased contractility of the CC induced by phenylephrine. Acetylcholine-induced relaxation was significantly reduced in SSS and SSK mice. Sodium nitroprusside-induced relaxation was higher in SSS mice compared to control and SSK mice. Nicotine-induced neurogenic and nitric oxide-dependent relaxation was significantly impaired in both SSS and SSK mice. An immunohistochemical analysis revealed co-localization of tyrosine hydroxylase and neuronal nitric oxide synthase-immunoreactive fibers in the CC. These findings highlight the complex nature of SS-related ED and suggest the limited efficacy of ketamine as a therapeutic intervention.

The Calcineurin-Drp1-Mediated Mitochondrial Fragmentation Is Aligned with the Differentiation of c-Kit Cardiac Progenitor Cells.

The heart contains a pool of c-kit+ progenitor cells which is believed to be able to regenerate. The differentiation of these progenitor cells is reliant on different physiological cues. Unraveling the underlying signals to direct differentiation of progenitor cells will be beneficial in controlling progenitor cell fate. In this regard, the role of the mitochondria in mediating cardiac progenitor cell fate remains unclear. Specifically, the association between changes in mitochondrial morphology with the differentiation status of c-kit+ CPCs remains elusive. In this study, we investigated the relationship between mitochondrial morphology and the differentiation status of c-kit+ progenitor cells. c-kit+ CPCs were isolated from 2-month-old male wild-type FVB mice. To activate differentiation, CPCs were incubated in α-minimal essential medium containing 10 nM dexamethasone for up to 7 days. To inhibit Drp1-mediated mitochondrial fragmentation, either 10 μM or 50 μM mdivi-1 was administered once at Day 0 and again at Day 2 of differentiation. To inhibit calcineurin, either 1 μM or 5 μM ciclosporin-A (CsA) was administered once at Day 0 and again at Day 2 of differentiation. Dexamethasone-induced differentiation of c-kit+ progenitor cells is aligned with fragmentation of the mitochondria via a calcineurin-Drp1 pathway. Pharmacologically inhibiting mitochondrial fragmentation retains the undifferentiated state of the c-kit+ progenitor cells. The findings from this study provide an alternative view of the role of mitochondrial fusion-fission in the differentiation of cardiac progenitor cells and the potential of pharmacologically manipulating the mitochondria to direct progenitor cell fate.

Intrinsic and synaptic properties of adult mouse spinoperiaqueductal gray neurons and the influence of neonatal tissue damage.

The periaqueductal gray (PAG) represents a key target of projection neurons residing in the spinal dorsal horn. In comparison to lamina I spinoparabrachial neurons, little is known about the intrinsic and synaptic properties governing the firing of spino-PAG neurons, or whether such activity is modulated by neonatal injury. In this study, this issue was addressed using ex vivo whole-cell patch clamp recordings from lamina I spino-PAG neurons in adult male and female FVB mice after hindpaw incision at postnatal day (P)3. Spino-PAG neurons were classified as high output, medium output, or low output based on their action potential discharge after dorsal root stimulation. The high-output subgroup exhibited prevalent spontaneous burst firing and displayed initial burst or tonic patterns of intrinsic firing, whereas low-output neurons showed little spontaneous activity. Interestingly, the level of dorsal root-evoked firing significantly correlated with the resting potential and membrane resistance but not with the strength of primary afferent-mediated glutamatergic drive. Neonatal incision failed to alter the pattern of monosynaptic sensory input, with most spino-PAG neurons receiving direct connections from low-threshold C-fibers. Furthermore, primary afferent-evoked glutamatergic input and action potential discharge in adult spino-PAG neurons were unaltered by neonatal surgical injury. Finally, Hebbian long-term potentiation at sensory synapses, which significantly increased afferent-evoked firing, was similar between P3-incised and naive littermates. Collectively, these data suggest that the functional response of lamina I spino-PAG neurons to sensory input is largely governed by their intrinsic membrane properties and appears resistant to the persistent influence of neonatal tissue damage.

Спонтанне посттравматичне відновлення рухової функції задньої кінцівки самців мишей

We studied the level of spontaneous post-traumatic recovery of motor function in male FVB mice. The left-side spinal cord hemisection was a model of damage. The locomotor activity and spasticity of the hindlimb were determined using the BBB and B scales and the Ashword scale during the first 12 weeks of the post-traumatic period. According to the results of the experimental study, at a later date in the post-traumatic period, the restoration of the function of the hind limb was observed at the level of 3.22 ± 0.46 points (out of 21 possible) on the BBB scale and 1.88 ± 0.27 points (out of 9 possible) on the B scale. Such indicators of hind limb locomotor activity conform to the phase of early recovery. However, at all studied time intervals after spinal cord injury, a consistently high level of hindlimb spasticity in experimental animals was noted – 3.17 ± 0.46 (out of 4 possible) on the Ashword scale.

Abstract 1336: Characterization of a novel transplantable model of obesity-driven basal-like breast cancer

Abstract Introduction: Breast cancer (BC) is a leading cause of cancer-related death in women, with the poorest prognosis found in the basal-like subtype. Although obesity is an established risk factor for basal-like BC, existing mouse models to study the interaction of basal-like BC and diet-induced obesity (DIO) are limited. The C3(1)/SV40 large T-antigen (TAg) transgenic mouse is an established, human-relevant basal-like mammary cancer model developed on an obesity-resistant FVB genetic background. The goal of this study was to create and characterize a transplantable C3TAg model on an obesity-sensitive C57BL/6J (B6) genetic background. Methods: Male C3TAg transgenic FVB mice were backcrossed with female wild-type B6 mice for 10 generations. Spontaneous mammary tumors from these female B6:C3TAg mice were dissociated and subcloned, generating 4 distinct cell lines differing in their expression of metabolic genes. A pilot orthotopic transplant study using the 4 candidate cell lines (at 3 cell concentrations of each line) in 96 B6 mice identified cell line 2.51 as an attractive model for further evaluation based on in vivo growth characteristics and initial genomic analyses. To assess the impact of DIO in this model, 61 female B6 mice were randomized to receive either control (n=30) or DIO (n=31) diet regimens for 24 weeks, then were orthotopically injected into the 4th mammary fat pad with cell line 2.51 (2.5x105 cells/mouse). The mice continued on their respective diets, were monitored for tumor development, and were euthanized 4 weeks post-injection. Tumors were collected, weighed and flash frozen for genomic, immunologic and biochemical analyses. Results: All 4 cell lines tested in the pilot study generated basal-like mammary tumors when orthotopically transplanted. Tumor gene expression profiling (via Affymetrix microarray) revealed inflammation and stem-like gene expression profiles discordant between cell lines tested, with tumors induced by cell line 2.51 showing striking enrichment in immune signatures relative to the other cell lines. DIO, relative to control diet, significantly accelerated transplanted 2.51 cell tumor progression, as indicated by increased tumor mass in mice from DIO relative to control diet-fed animals. Genomic, biochemical, and immunological assessment of the tumor-enhancing effects of DIO in this model are ongoing. Conclusions: Herein, we report the development and characterization of a novel transplantable C3TAg mammary tumor model in C57BL/6J mice, highly responsive to energy balance. This work represents a promising new tool for preclinical studies of the interactions between diet, obesity, immunity and basal-like breast cancer. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Meredith S. Carson, Elaine M. Glenny, Violet A. Kiesel, Ashlee Taylor, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Michael F. Coleman, Stephen D. Hursting. Characterization of a novel transplantable model of obesity-driven basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1336.

Complex II‐linked mitochondrial respiration is upregulated during postnatal development in high‐altitude grown mice

Recently, we have shown that chronic normobaric hypoxia (12% O2 – 21 days) triggers mitochondrial and cell‐ metabolic plasticity in the retrosplenial cortex of male adult FVB mice but not in SD rats. In these animals we reported a transient upregulation of anaerobic metabolic pathways (glycolysis + lactate metabolism) followed by an attenuation in the mitochondrial respiration. These results pointed to an optimized use of oxygen at cellular level in an area of the brain responsible of processing visual information and spatial memory. Features of great importance in the biological fitness. In the current work we investigate how the residence in high altitude (La Paz – Bolivia, 3,600 m) affects the developmental profile of the mitochondrial respiratory efficiency in the retrosplenial cortex of mice and rats. We hypothesize that the mitochondrial plasticity observed in adult mice will have a process of maturation during postnatal development. This response shall be absent in rats.To do so, we measured the mitochondrial oxygen consumption rate (OCR) in saponin‐permeabilized brain samples from FVB mice and SD rats born and grown in La Paz. The animals were sacrificed at postnatal ages p7, p14, p21, and adulthood (P90), and the OCR linked to the use of NADH (N pathway), FADH2 (S pathway), or both (NS pathway) was quantified by means of high‐resolution respirometry (OROBOROS).Our results show a pattern of maturation (increase) of the mitochondrial respiration in the retrosplenial cortex along with the postnatal development (21 days+) of mice and rats. However, an upregulation of the participation of the S pathway (complex II‐linked) in the mitochondrial respiration was observed during the first three weeks of age only in mice. This observation supports previous works suggesting a key role of the complex II and succinate (substrate of complex II) in the successful cellular and mitochondrial acclimatization to hypoxia. These findings contribute to further establish FVB mice and SD rats as a model to study divergent acclimatization to hypoxia.

Sex Differences in Glomerular Protein Expression and Effects of Soy-Based Diet on Podocyte Signaling.

Background:Kidney disease is a major public health issue arising from loss of glomerular podocyte function, and there are considerable sex differences in its prognosis. Evidence suggests a renoprotective effect of estrogen and soy diet-derived phytoestrogens, although the molecular basis for this is poorly understood.Objective:Here, we aim to assess sex differences in expression of key proteins associated with podocyte survival and determine the effects of dietary soy on glomerular and podocyte signaling.Methods:Male and female FVB mice were fed control, low (1%), and high (20%) doses of isolated soy protein (ISP) in utero and until 100 days of age. Spot urine was collected to measure proteinuria and isolated glomeruli were used to quantify activated and total levels of nephrin, Akt, and ERK1/2. To investigate protective effects of specific soy phytoestrogens, cultured podocytes were treated with or without daidzein and subject to control or high glucose as a model of podocyte injury.Results:Nephrin and Akt were elevated at baseline in glomeruli from females compared to males. Both sexes that were fed 1% and 20% ISP displayed robust increases in total glomerular Akt compared to controls, and these effects were more prominent in females. A similar trend at both doses in both sexes was observed with activated Akt and total nephrin. Notably, males exclusively showed increased phosphorylation of nephrin and extracellular signal-regulated kinase (ERK) at the 1% ISP dose; however, no overt changes in urinary albumin excretion or podocin levels were observed, suggesting that the soy diets did not impair podocyte function. Finally, in cultured male and female podocytes, daidzein treatment suppressed high glucose-induced ERK activation.Conclusions:Together, our findings reveal a putative mechanism to explain the protective influence of sex on kidney disease progression, and they provide further evidence to support a beneficial role for dietary soy in preserving glomerular function.

CD-1 Outbred Mice Produce Less Variable Ultrasonic Vocalizations Than FVB Inbred Mice, While Displaying a Similar Developmental Trajectory.

The production of ultrasonic vocalizations (USVs) in neonatal mice is a critical means of communication that is used to elicit maternal care. Alterations in neonatal USV production is also an indicator of neurological deficits. However, USVs have been predominately assessed in inbred animals and are significantly understudied in outbred mice, even though outbred animals better represent the genetic diversity of humans and are used in several neurological disorder models. To determine the reproducibility of USVs across models, we compared male and female CD-1 (outbred) and FVB (inbred) mice on postnatal days (PD) 4, 8, 12, 16, and 20. We found that CD-1 and FVB mice displayed a similar developmental trajectory of USVs. However, CD1 mice emitted more USVs on PD 12 than FVB mice. In addition, FVB mice emitted a longer duration of calls on PD 4 and 8 and a higher overall maximum and minimum frequency of USVs than CD-1 mice. No differences in mean amplitude were found between groups. We also detected numerous significant differences between outbred and inbred mice when comparing each group's call composition. We next assessed the relative variability of mouse vocalizations between groups, finding that outbred mice were less variable than inbred mice. For the spectral and temporal characteristics of the USVs, variability was similar between groups. Altogether, we found that CD-1 outbred mice display a similar, if not lower, degree of variability than FVB inbred mice when assessing neonatal USVs.

Triggering mitochondrial plasticity in the liver of rodents is key for successful acclimatization to hypoxia

Liver is the most metabolically active organ in mice and rats, accounting for up to 50% of the body's energy expenditure. As such, this organ is highly sensitive to the reduction in O2 availability occurring in hypoxic conditions such as high-altitude. Keeping in mind that mice, but not rats, successfully colonized habitats over 2,500 masl, we showed previously that mice only increase their metabolic rates under chronic hypoxia. Accordingly, we hypothesized here that mice and rats have divergent mitochondrial and molecular features in the liver that determine an adequate energy balance in mice (not rats) during the process of acclimatization to hypoxia. To test this hypothesis, we used saponin-permeabilized samples of liver from male adult FVB mice and SD rats after exposure to hypoxia (12% O2, for 0, 1, 7 or 21 days) to measure the mitochondrial O2 consumption rate (OCR) and the activation of the N (complex I-linked), S (complex II-linked), and NS (complex I&II-linked) electron-transfer pathways in the high-resolution respirometer O2K (OROBOROS instruments). Additional experiments were performed to evaluate the activity of representative enzymes from the glycolytic (hexokinase [HK]), aerobic (pyruvate dehydrogenase [PDH]), and anaerobic (lactate dehydrogenase [LDH]) metabolic pathways by spectrometric methods. Our results show that liver mitochondria in mice increase the OCR and improve the activation of the electron-transfer pathways S (complex II-linked) and NS (complexes I&II-linked). in line, the glycolytic and aerobic metabolic enzymatic activities (HK and PDH) increased, while the anaerobic metabolism (LDH) reduced after seven-days hypoxia. On the other hand, rats were shown to be unable of altering liver mitochondrial respiration but augmented glycolytic and anaerobic metabolic activities (HK and LDH) and concomitantly reduced the aerobic metabolism (PDH). These results show that liver mitochondria of mice display plastic responses in the utilization of oxygen during the process of acclimatization to hypoxia and that these changes are supported by adjustments in the glycolytic, aerobic, and anaerobic metabolic pathways. Contrastingly, together with the inability to adjust their mitochondrial activity, rats depend heavily on anaerobic pathways (known to be less effective) to produce energy. Our results contribute to explain why mice, but not rats, are present in high-altitude environments.

Mice and Rats Display Different Ventilatory, Hematological, and Metabolic Features of Acclimatization to Hypoxia.

Phylogeographic studies showed that house mice (Mus musculus) originated in the Himalayan region, while common rats (Rattus rattus and Rattus norvegicus) come from the lowlands of China and India. Accordingly, it has been proposed that its origins gave mice, but not rats, the ability to invade ecological niches at high altitudes (pre-adaptation). This proposal is strongly supported by the fact that house mice are distributed throughout the world, while common rats are practically absent above 2,500 m. Considering that the ability of mammals to colonize high-altitude environments (>2,500 m) is limited by their capability to tolerate reduced oxygen availability, in this work, we hypothesize that divergences in the ventilatory, hematological, and metabolic phenotypes of mice and rats establish during the process of acclimatization to hypoxia (Hx). To test this hypothesis male FVB mice and Sprague-Dawley (SD) rats were exposed to Hx (12% O2) for 0 h (normoxic controls), 6 h, 1, 7, and 21 days. We assessed changes in ventilatory [minute ventilation (VE), respiratory frequency (fR), and tidal volume (VT)], hematological (hematocrit and hemoglobin concentration), and metabolic [whole-body O2 consumption (VO2) and CO2 production (VCO2), and liver mitochondrial oxygen consumption rate (OCR) parameters]. Compared to rats, results in mice show increased ventilatory, metabolic, and mitochondrial response. In contrast, rats showed quicker and higher hematological response than mice and only minor ventilatory and metabolic adjustments. Our findings may explain, at least in part, why mice, but not rats, were able to colonize high-altitude habitats.

β2-Adrenergic Signaling Modulates Mitochondrial Function and Morphology in Skeletal Muscle in Response to Aerobic Exercise.

The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that β2-adrenergic receptor (β2-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under β2-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by β2-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. β2-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by β2-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective β-AR agonist isoproterenol (ISO) in the presence of the specific β2-AR antagonist ICI or during protein kinase A (PKA) and Gαi protein blockade. Our in vitro data show that β-AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on β2-AR activation through a Gαs-PKA signaling cascade. In conclusion, we provide evidence for AE-induced β2-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. β2-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.

Abstract 3966: The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. Our current study examines whether moderate weight loss or treatment with the non-steroidal anti-inflammatory drug sulindac suppresses the tumor-promoting effects of obesity in a mouse model of colon cancer. Methods: Male FVB mice were treated with azoxymethane (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) diet. After 15 weeks, there was an interim sacrifice of 5 mice/group. The DIO mice were then further randomized to remain on DIO or switch to control diet to induce weight loss and become formerly obese mice (FOB), and within each diet group (control, DIO, and FOB), half the mice were randomized to start sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were euthanized. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P&amp;lt;0.05), while FOB mice had intermediate levels of adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had 10-fold greater tumor multiplicity relative to control mice (P=0.07). At study endpoint, DIO mice had greater tumor multiplicity compared to both control (P&amp;lt;0.05) and FOB (P&amp;lt;0.01) mice, which did not significantly differ from each other. Sulindac reduced tumor multiplicity in both control (P&amp;lt;0.05) and DIO (P&amp;lt;0.0001) mice, but not FOB mice. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus control (P&amp;lt;0.05) and DIO+sulindac (P&amp;lt;0.01) mice, but not in comparison to FOB mice. Gene expression in paired colon tumor and mesenteric adipose tissue samples was assessed by microarray. Ingenuity pathway analysis (IPA) of the significant (P&amp;lt;0.05) differentially expressed tumor genes indicated that both weight loss and sulindac reduced signaling related to matrix metalloproteinases (MMPs) and inflammation. In the visceral adipose tissue, IPA demonstrated that weight loss reduced signaling related to B cell receptors and glucose/fatty acid metabolism while sulindac decreased MMP, prostaglandin, and PPAR signaling. Additional analyses to explore the potential role of cross-talk between these two tissues are being performed, and key findings will be validated in the tissues at the protein level and via mechanistic cell culture experiments. Conclusions: Both moderate weight loss and sulindac treatment completely reverse the effects of chronic obesity on colon tumorigenesis, the latter independent of any change in adiposity. Our findings suggest that an investigation regarding the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted, particularly for those unable to achieve moderate weight loss. Citation Format: Laura W. Bowers, Arunima Punjala, Alison Q. Jeffries, Stephanie A. Montgomery, Andrew J. Dannenberg, Stephen D. Hursting. The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3966.

Abstract 2262: Low-dose targeted radionuclide therapy has favorable local and systemic effects on immune populations in a murine prostate cancer model

Abstract Introduction: Prostate cancer remains the second leading cause of cancer-related death in American men because of its metastatic, incurable form. Prostate cancer responds poorly to checkpoint blockade likely due to its immunosuppressive microenvironment with relatively few infiltrating CD8+ T cells, limited mutational burden, and activity of myeloid-derived suppressor cells (MDSCs). External beam radiotherapy (EBRT) has been shown to help overcome this suppressive state, but EBRT is infeasible for patients with widely metastatic disease. Our group has developed a compound called NM600 that can be used to deliver radiation to all sites of disease simultaneously in an approach called Targeted radionuclide therapy (TRT). However, the effects of TRT on immune populations within prostate tumors are not yet well described. In this study, we examined the effects of TRT in the form of 90Y-NM600 on mice bearing Myc-CaP prostate tumors. Methods: 6-week old male FVB mice were implanted subcutaneously with Myc-CaP cells, then given a single intravenous injection of either 50 (“low-dose”) or 250 (“high-dose”) μCi of 90Y-NM600, estimated to deliver 3.1 Gy or 15.5 Gy to 300 mm3 tumors, respectively. Groups of mice (n=3) were then euthanized at several timepoints following TRT administration. Their tumors and spleens were collected for analysis by flow cytometry and Luminex cytokine analysis. A separate group (n=5) were followed for survival to 2000 mm3. Results: Flow cytometry of splenocytes revealed that high-dose, but not low-dose TRT caused a 6-fold increase in numbers of MDSCs (p = 0.002) at Day 21 compared to baseline. Additionally, low-dose TRT caused a 2-fold (p = 0.005) increase in CD8+ T cells at Day 21 compared to baseline, unlike in the high-dose condition. These CD8+ T cells in the low-dose TRT condition were predominantly proliferating naïve T cells. Flow cytometry of tumor infiltrating lymphocytes showed a 13% increase in CD8+ T cells in both dose conditions at Day 14 (p = 0.03). Returning CD8+ T cells displayed high expression of PD-1, CTLA-4, and LAG-3. Within the tumor, there was an 8-fold increase in chemokines including CXCL1, Rantes, and MIP2 at Day 21, suggesting that there may be further recruitment of tumor infiltrating cells even three weeks following treatment. Mice treated with high-dose TRT (median survival 37 days, p = .029) had significantly improved median survival compared to control (median survival 23 days), unlike low-dose treated mice (median survival 29 days, p =.250). Conclusions: These data suggest that low-dose TRT is superior for immunomodulation because, although it has little effect on tumor growth, it increases intratumoral CD8+ T cell infiltration while avoiding deleterious systemic immune effects. We hypothesize that immunomodulatory TRT combined with checkpoint blockade will improve anti-tumor efficacy compared to high-dose TRT. Citation Format: Hemanth Kumar Potluri, Reinier Hernandez, Christopher D. Zahm, Joseph Grudzinski, Christopher Massey, Jamey Weichert, Douglas G. McNeel. Low-dose targeted radionuclide therapy has favorable local and systemic effects on immune populations in a murine prostate cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2262.

PDE5 inhibitor sildenafil attenuates cardiac microRNA 214 upregulation and pro-apoptotic signaling after chronic alcohol ingestion in mice.

Abusive chronic alcohol consumption can cause metabolic and functional derangements in the heart and is a risk factor for development of non-ischemic cardiomyopathy. microRNA 214 (miR-214) is a molecular sensor of stress signals that negatively impacts cell survival. Considering cardioprotective and microRNA modulatory effects of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, we investigated the impact of chronic alcohol consumption on cardiac expression of miR-214 and its anti-apoptotic protein target, Bcl-2 and whether sildenafil attenuates such changes. Adult male FVB mice received unlimited access to either normal liquid diet (control), alcohol diet (35% daily calories intake), or alcohol + sildenafil (1mg/kg/day, p.o.) for 14weeks (n = 6-7/group). The alcohol-fed groups with or without sildenafil had increased total diet consumption and lower body weight as compared with controls. Echocardiography-assessed left ventricular function was unaltered by 14-week alcohol intake. Alcohol-fed group had 2.6-fold increase in miR-214 and significant decrease in Bcl-2 expression, along with enhanced phosphorylation of ERK1/2 and cleavage of PARP (marker of apoptotic DNA damage) in the heart. Co-ingestion with sildenafil blunted the alcohol-induced increase in miR-214, ERK1/2 phosphorylation, and maintained Bcl-2 and decreased PARP cleavage levels. In conclusion, chronic alcohol consumption triggers miR-214-mediated pro-apoptotic signaling in the heart, which was prevented by co-treatment with sildenafil. Thus, PDE5 inhibition may serve as a novel protective strategy against cardiac apoptosis due to chronic alcohol abuse.

Modulation of CYP3A Activity to Increase the Oral Bioavailability of Ibrutinib

BackgroundIbrutinib (Imbruvica; PCI‐32765), an orally administered inhibitor of Bruton’s tyrosine kinase, is considered a breakthrough targeted therapy that is approved as frontline therapy in chronic lymphocytic leukemia (CLL). Ibrutinib has an average oral bioavailability in humans of &lt;4% with substantial variability in exposure, which predisposes patients to unpredictable and potentially harmful adverse events such as bleeding and atrial fibrillation. As ibrutinib is subject to extensive first‐pass metabolism by CYP3A following oral administration, inhibition of CYP3A activity may be a promising strategy to increase the bioavailability and decrease inter‐individual pharmacokinetic (PK) variability of ibrutinib. The objective of this study was to characterize the impact of CYP3A on the PK of ibrutinib and its main active metabolite, PCI‐45227, in mice.MethodsTo characterize the impact of CYP3A on ibrutinib disposition, in vivo PK studies were performed in wild‐type (WT) male and female FVB mice treated with CYP3A inhibitors including ketoconazole (50 mg/kg p.o.) and cobicistat (30 mg/kg; p.o.) or the respective vehicle controls (PEG400 or corn oil p.o.) thirty minutes prior to dosing with ibrutinib (10 mg/kg p.o.). Based on these results, CYP3A(−/−) female FVB mice were treated with ibrutinib (10 mg/kg p.o. or 1 mg/kg i.v.) alone or thirty minutes after administration of cobicistat (30 mg/kg; p.o.). Concentrations of ibrutinib and PCI‐45227 in plasma were determined by a validated method based on liquid chromatography‐tandem mass spectrometry, and PK parameter estimates were calculated with the software package Phoenix WinNonlin (Version 8.1). Student t‐tests comparing mean AUC values between groups were used to determine significance.ResultsThe peak plasma concentration (Cmax) and area under the concentration‐time curve (AUC) of ibrutinib were ~10‐fold higher in mice administered pharmacologic CYP3A inhibitors prior to ibrutinib (P &lt; 0.0001) with a substantial decrease in the PCI‐45227 to ibrutinib AUC ratio. As expected, when ibrutinib was administered orally to CYP3A(−/−) mice, the AUC of ibrutinib in CYP3A(−/−) mice was increased ~10‐fold (P &lt; 0.0001) with almost no formation of the PCI‐45227 metabolite compared with WT mice. Cobicistat did not impact ibrutinib AUC in CYP3A(−/−) mice (P &gt; 0.9), which suggests that CYP3A inhibition is the major mechanism for cobicistat‐induced increases in ibrutinib exposure.ConclusionsPreviously obtained data with other kinase inhibitors indicate that patients with high CYP3A4 activity may benefit from an increased dose to achieve drug concentrations required to interact with kinase targets; a strategy by which enzyme activity is inhibited could achieve similar results. In view of the established inverse‐correlation between absolute bioavailability and inter‐individual PK variability, the current data provide a rationale for the development of exploratory clinical studies aimed at decreasing the variability in ibrutinib exposure by concomitant administration of CYP3A4 inhibitors. Future preclinical efficacy studies will determine the impact of this strategy on survival in mouse models of B‐cell malignancies.

Mice but not rats modulate liver mitochondrial machinery in chronic hypoxia

Mice (FVB) permanently living at high‐altitude have increased respiratory frequencies and sustained or increased metabolic rates. On the other hand, rats permanently living at high‐altitude (SD) have impaired respiratory control, high hematocrit and hemoglobin, signs of pulmonary hypertension, and reduced metabolism; all these considered to be detrimental. This is coincident with the fact that common rats are not found in natural high‐altitude environments. At sea level, we reported that mice, but nor rats, exposed to 21 days of hypoxia (12% O2) increase their metabolic rate (O2 consumption and CO2 production), similarly to other rodent species that are considered well adapted to high‐altitude. As mitochondria is the final user of O2 in metabolism, we were interested in the role of mitochondria behind this divergence in physiological adjustments. We used saponin‐permeabilized samples of liver (most energy‐consuming organ in rodents), from male adult FVB mice and SD rats after exposure to hypoxia (12% O2, for 0, 1, 7 or 21 days), to measure the mitochondrial O2 consumption rate (OCR) following the SUIT‐01 protocol in the high‐resolution respirometer O2K (OROBOROS instruments). OCR was measured with substrates activating the NADH, succinate , and fatty acid pathways of the electron transport chain (ETC). We also measured the maximum capacity of ETC by uncoupling the electron transport from the oxidative phosphorylation with CCCP. The level of activation of complexes I and II was reported as the OCR for the N or S pathway correspondingly, divided by the maximum capacity (flux control ratio).Our preliminary results show that, compared to rats, mice have significantly higher ETC maximum capacities in normoxia and hypoxia. Moreover, after 21 days of hypoxia, mice showed an increase of 43% in maximum capacity in comparison to normoxic controls, while no increase in maximum capacity was evidenced in rats. In mice, we observed that complex I is transiently hyperactivated after 1 day of hypoxia and then, its activity reduces down below control levels by day 21 of hypoxia. Contrastingly, complex II activity is reduced after one day of hypoxia, getting back to control levels by day 21 of hypoxia. Furthermore, in rats, an increased activity of complex I occurred after 21 days of hypoxia. Regardless of normoxic or hypoxic exposure, rats showed higher activation levels of complex II than mice.These results show that mice, but not rats, have the ability to modulate their liver metabolic mitochondrial machinery under chronic hypoxia. The higher and adjustable maximum capacity in mice, suggest a more powerful and plastic liver mitochondrial machinery than in rats. This might explain in part their ability to keep sustained metabolic rates under hypoxic conditions.Support or Funding InformationSupported by NSERC, the Health Respiratory Network of Québec and CIHR.